DNAs encoding mammalian histamine receptor of the H4 subtype and encoded protein

ABSTRACT

DNAs encoding the mammalian histamine H4 receptors have been cloned and characterized. These recombinant molecules are capable of expressing biologically active histamine H4 receptor protein. The cDNA&#39;s have been expressed in recombinant host cells that produce active recombinant protein. The pharmacology of known histamine ligands is demonstrated. The recombinant protein may be purified from the recombinant host cells. In addition, recombinant host cells are utilized to establish methods to identify modulators of the receptor activity, and receptor modulators are identified.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of U.S. patent application Ser. No. 09/790,849,filed Feb. 22, 2001, now abandoned, which claims benefit of U.S.Provisional Application Ser. No. 60/208,260, filed May 31, 2000, nowexpired, the contents of both applications being incorporated in theirentireties herein by reference.

BACKGROUND OF THE INVENTION

Histamine is a multifunctional chemical transmitter that signals throughcell surface receptors that are linked to intracellular pathways viaguanine nucleotide binding proteins. This class of cell surface receptoris called G-protein coupled receptors or GPCRs. There are currentlythree subtypes of histamine receptors that have been definedpharmacologically and have been divided into H1, H2, and H3classifications (Hill, et al. 1997). The H1 histamine receptor has beencloned (Yamashita, et al. 1991) and is the target of drugs such asdiphenhydramine to block the effects of histamine on smooth muscle inallergic responses. The H2 histamine receptor has been cloned (Gantz etal. 1991) and is the target of drugs such as ranitidine to block theeffects of histamine on acid secretion in the stomach. The H3 histaminereceptor, which was hypothesized to exist in 1983 (Arrang, et al. 1983),has been cloned (Lovenberg et al., 1999) and is currently a target fordevelopment of central nervous system drugs. There are numerousadditional functions of histamine in humans which may be mediated byhistamine receptors of unknown class. For example, histamine is achemotactic factor for leukocytes, causing their accumulation in areasof allergic challenge such as skin, nose, eyes and lungs (De Vos, 1999).The receptor responsible for mediating this effect of histamine is notknown.

The present invention relates to the isolation and characterization ofmammalian cDNAs encoding a novel histamine receptor (histamine H4receptor) and the uses thereof.

SUMMARY OF THE INVENTION

DNA molecules encoding a mammalian histamine H4 receptor have beencloned and characterized and represent a novel member of the class ofreceptors that couple to G-proteins. Using a recombinant expressionsystem, functional DNA molecules encoding these histamine H4 receptorshave been isolated from mouse, rat, guinea pig, and human. Thebiological and structural properties of these proteins are disclosed, asis the amino acid and nucleotide sequence. The recombinant protein isuseful for a variety of purposes, including but not limited toidentifying modulators of the human histamine H4 receptor. The histamineH4 receptors of mouse, rat, and guinea pig have a variety of uses,including but not limited to resolving pharmacological differencesobserved between different mammalian species, particularly since guineapig, rat, and murine species are commonly used in pre-clinicalevaluation of new chemical entities. Such modulators can includeagonists, antagonists, and inverse agonists. Modulators identified inthe assays disclosed herein are useful, for example, as therapeuticagents, prophylactic agents, and diagnostic agents. Indications for saidtherapeutic agents include, but are not limited to, asthma, allergy,inflammation, cardiovascular and cerebrovascular disorders, non-insulindependent diabetes mellitus, hyperglycemia, constipation, arrhythmia,disorders of the neuroendocrine system, stress, and spasticity, as wellas acid secretin, ulcers, airway constriction, and prostate dysfunction.The recombinant DNA molecules, and portions thereof, have a variety ofuses including but not limited to isolating homologues of the DNAmolecules, identifying and isolating genomic equivalents of the DNAmolecules, and identifying, detecting or isolating mutant forms of theDNA molecules.

BRIEF DESCRIPTION OF THE DRAWING DRAWINGS

FIG. 1—The complete nucleotide coding sequence of human histamine H4receptor (SEQ ID NO: 1) including untranslated regions is shown.

FIG. 2—The amino acid sequence of human histamine H4 receptor (SEQ IDNO: 2) is shown.

FIG. 3—The tissue distribution of the human histamine H4 receptor isshown.

FIG. 4—Binding of [³H]-histamine to the human H4 receptor is shown.

FIG. 5 Panels A, B and C—The complete nucleotide coding sequence ofmouse (A), guinea pig (B), and rat (C) histamine H4 receptors is shown.

FIG. 6 Panels A, B and C—The amino acid sequence of mouse (A) (SEQ IDNO: 5), guinea pig (B) (SEQ ID NO: 7), and rat (C) (SEQ ID NO: 6)histamine H4 receptors is shown.

FIG. 7—The alignment of the polynucleotide sequences of the human,guinea pig, mouse and rat histamine H4 receptor is shown.

FIG. 8—The alignment of the polypeptide sequences of the human, guineapig (SEQ ID NO: 10), mouse (SEQ ID NO: 8) and rat (SEQ ID NO: 9)histamine H4 receptor is shown.

DETAILED DESCRIPTION

The present invention relates to DNA encoding human histamine H4receptors that was isolated from a cDNA library from human bone marrow.The human histamine H4 receptor, as used herein, refers to protein whichcan specifically function as a receptor for histamine of the H4subclass.

The present invention also relates to DNA molecules encoding mammalianhistamine H4 receptors. In particular, the present invention relates toDNA molecules encoding guinea pig (cavia porcellus), rat (rattusrattus), and murine (mus musculus) histamine H4 receptors. The termmammalian histamine H4 receptor, as used herein, refers to protein whichcan specifically function as a receptor for histamine of the H4subclass.

The complete or partial amino acid sequence of human, guinea pig, rat,or murine histamine H4 receptor was not previously known, nor was thecomplete or partial nucleotide sequence encoding histamine H4 receptorknown. It is predicted that a wide variety of cells and cell types willcontain the described mammalian histamine H4 receptor. Vertebrate cellscapable of producing histamine H4 receptor include, but are not limitedto histamine H4 receptor expressing cells isolated from cells that showsensitivity to or bind histamine. Such cells can be derived from bonemarrow, spleen, blood, or other tissues.

Other cells and cell lines may also be suitable for use to isolatehistamine H4 receptor cDNA. Selection of suitable cells may be done byscreening for ³[H]histamine binding or inhibition of adenylate cyclasein response to histamine. Histamine H4 receptor activity can bemonitored by performing a ³[H]-histamine binding assay (see experimentalsection) or by direct measurement of inhibition of adenylate cyclase dueto histamine H4 receptor activation or by incorporation of GTP-gamma-S(Clark, Korte et al. 1993). Cells that possess histamine H4 receptoractivity in this assay may be suitable for the isolation of histamine H4receptor DNA or mRNA.

Any of a variety of procedures known in the art may be used to clonemammalian histamine H4 receptor DNA. These methods include, but are notlimited to, direct functional expression of the histamine H4 receptorgenes following the construction of a histamine H4 receptor-containingcDNA library in an appropriate expression vector system. Another methodis to screen histamine H4 receptor-containing cDNA library constructedin a bacteriophage or plasmid shuttle vector with a labelledoligonucleotide probe designed from the amino acid sequence of the humanhistamine H4 receptor. An additional method consists of screening ahistamine H4 receptor-containing cDNA library constructed in abacteriophage or plasmid shuttle vector with a partial cDNA encoding thehistamine H4 receptor protein. This partial cDNA is obtained by thespecific PCR amplification of human histamine H4 receptor DNA fragmentsthrough the design of degenerate oligonucleotide primers from the aminoacid sequence of the purified histamine H4 receptor protein, describedherein.

Another method is to isolate RNA from histamine H4 receptor-producingcells and translate the RNA into protein via an in vitro or an in vivotranslation system. The translation of the RNA into a peptide or aprotein will result in the production of at least a portion of thehistamine H4 receptor protein that can be identified by, for example,immunological reactivity with an anti-histamine H4 receptor antibody orby biological activity of histamine H4 receptor protein. In this method,pools of RNA isolated from histamine H4 receptor-producing cells can beanalyzed for the presence of an RNA which encodes at least a portion ofthe histamine H4 receptor protein. Further fractionation of the RNA poolcan be done to purify the histamine H4 receptor RNA from non-histamineH4 receptor RNA. The peptide or protein produced by this method may beanalyzed to provide amino acid sequences which in turn are used toprovide primers for production of histamine H4 receptor cDNA, or the RNAused for translation can be analyzed to provide nucleotide sequencesencoding histamine H4 receptor and produce probes for this production ofhistamine H4 receptor cDNA. This method is known in the art and can befound in, for example, Maniatis, T., Fritsch, E. F., Sambrook, J. inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. 1989.

It is readily apparent to those skilled in the art that other types oflibraries, as well as libraries constructed from other cells or celltypes, may be useful for isolating histamine H4 receptor-encoding DNA.Other types of libraries include, but are not limited to, cDNA librariesderived from other cells, from organisms other than human, and genomicDNA libraries that include YAC (yeast artificial chromosome) and cosmidlibraries.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have histamineH4 receptor activity. The selection of cells or cell lines for use inpreparing a cDNA library to isolate histamine H4 receptor cDNA may bedone by first measuring cell associated histamine H4 receptor activityusing the measurement of histamine H4 receptor-associated biologicalactivity or a ³H-histamine binding assay or any radioligand bindinginvolving a ligand that has the ability to bind to the histamine H4receptor.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in Maniatis, T., Fritsch, E. F., Sambrook, J.,Molecular Cloning: A Laboratory Manual, Second Edition (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989).

It is also readily apparent to those skilled in the art that DNAencoding histamine H4 receptor may also be isolated from a suitablegenomic DNA library. Construction of genomic DNA libraries can beperformed by standard techniques well known in the art. Well knowngenomic DNA library construction techniques can be found in Maniatis,T., Fritsch, E. F., Sambrook, J. in Molecular Cloning: A LaboratoryManual, Second Edition (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989).

Other mammalian histamine H4 receptor cDNAs may be isolated byconducting PCR or RACE using identical or degenerate primers designedbased on the sequences of guinea pig, rat, murine, or human histamine H4receptor. PCR products are isolated and the sequence is determined andcompared to the histamine H4 receptor sequences described herein toestablish the identity of other mammalian histamine H4 receptor cDNAs.

In order to clone the histamine H4 receptor gene by the above methods,the amino acid sequence of histamine H4 receptor may be necessary. Toaccomplish this, histamine H4 receptor protein may be purified andpartial amino acid sequence determined by automated sequencers. Thepredicted amino acid sequence of human, guinea pig, rat, and murinehistamine H4 receptors is described herein, and may also be used toassist in cloning the histamine H4 gene. It is not necessary todetermine the entire amino acid sequence, but the linear sequence of tworegions of about 6 to 8 amino acids from the protein is determined forthe production of primers for PCR amplification of a partial humanhistamine H4 receptor DNA fragment.

Once suitable amino acid sequences have been identified, the DNAsequences capable of encoding them are synthesized. Because the geneticcode is degenerate, more than one codon may be used to encode aparticular amino acid, and therefore, the amino acid sequence can beencoded by any of a set of similar DNA oligonucleotides. Only one memberof the set will be identical to the histamine H4 receptor sequence butwill be capable of hybridizing to histamine H4 receptor DNA even in thepresence of DNA oligonucleotides with mismatches. The mismatched DNAoligonucleotides may still sufficiently hybridize to the histamine H4receptor DNA to permit identification and isolation of histamine H4receptor encoding DNA. DNA isolated by these methods can be used toscreen DNA libraries from a variety of cell types, from invertebrate andvertebrate sources, and to isolate homologous genes.

Purified biologically active histamine H4 receptor may have severaldifferent physical forms. Histamine H4 receptor may exist as afull-length nascent or unprocessed polypeptide, or as partiallyprocessed polypeptides or combinations of processed polypeptides. Thefull-length nascent histamine H4 receptor polypeptide may bepost-translationally modified by specific proteolytic cleavage eventswhich result in the formation of fragments of the full length nascentpolypeptide. One example of this is the cleavage of the signal peptideafter translation into the endoplasmic reticulum. A fragment, orphysical association of fragments may have the full biological activityassociated with histamine H4 receptor however, the degree of histamineH4 receptor activity may vary between individual histamine H4 receptorfragments and physically associated histamine H4 receptor polypeptidefragments.

The cloned histamine H4 receptor DNA obtained through the methodsdescribed herein may be recombinantly expressed by molecular cloninginto an expression vector containing a suitable promoter and otherappropriate transcription regulatory elements, and transferred intoprokaryotic or eukaryotic host cells to produce recombinant histamine H4receptor protein. Techniques for such manipulations are fully describedin Maniatis, T, et al., supra, and are well known in the art.

Expression vectors are defined herein as DNA sequences that are requiredfor the transcription of cloned copies of genes and the translation oftheir mRNAs in an appropriate host. Such vectors can be used to expresseukaryotic genes in a variety of hosts such as bacteria including E.coli, blue-green algae, plant cells, insect cells, fungal cellsincluding yeast cells, and animal cells.

Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal cells or bacteria-fungal cellsor bacteria-invertebrate cells. An appropriately constructed expressionvector should contain: an origin of replication for autonomousreplication in host cells, selectable markers, a limited number ofuseful restriction enzyme sites, a potential for high copy number, andactive promoters. A promoter is defined as a DNA sequence that directsRNA polymerase to bind to DNA and initiate RNA synthesis. A strongpromoter is one that causes mRNAs to be initiated at high frequency andoptimally does not greatly limit the proliferation of the host.Expression vectors may include, but are not limited to, cloning vectors,modified cloning vectors, specifically designed plasmids or viruses.

A variety of mammalian expression vectors may be used to expressrecombinant human histamine H4 receptor in mammalian cells. Commerciallyavailable mammalian expression vectors which may be suitable forrecombinant mammalian histamine H4 receptor expression, include but arenot limited to, pMAMneo (Clontech), pcDNA3 (Invitrogen), pMC1neo(Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), pCIneo (Promega),EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12)(ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr(ATCC 37146), pUCTag (ATCC 37460), and IZD35 (ATCC 37565).

A variety of bacterial expression vectors may be used to expressrecombinant mammalian histamine H4 receptor in bacterial cells.Commercially available bacterial expression vectors which may besuitable for recombinant mammalian histamine H4 receptor expressioninclude, but are not limited to pET vectors (Novagen) and pQE vectors(Qiagen).

A variety of fungal cell expression vectors may be used to expressrecombinant mammalian histamine H4 receptor in fungal cells such asyeast. Commercially available fungal cell expression vectors which maybe suitable for recombinant mammalian histamine H4 receptor expressioninclude but are not limited to pYES2 (Invitrogen) and Pichia expressionvector (Invitrogen).

A variety of insect cell expression vectors may be used to expressrecombinant mammalian histamine H4 receptor in insect cells.Commercially available insect cell expression vectors which may besuitable for recombinant expression of mammalian histamine H4 receptorinclude but are not limited to pBlueBacII (Invitrogen).

DNA encoding mammalian histamine H4 receptor may be cloned into anexpression vector for expression in a recombinant host cell. Recombinanthost cells may be prokaryotic or eukaryotic, including but not limitedto bacteria such as E. coli, fungal cells such as yeast, mammalian cellsincluding but not limited to cell lines of human, bovine, porcine,monkey and rodent origin, and insect cells including but not limited todrosophila and silkworm derived cells. Cell lines derived from mammalianspecies which may be suitable and which are commercially available,include but are not limited to, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92),NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616),BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), L-cells, and HEK-293 (ATCCCRL1573).

The expression vector may be introduced into host cells via any one of anumber of techniques including but not limited to transformation,transfection, protoplast fusion, lipofection, and electroporation. Theexpression vector-containing cells are clonally propagated andindividually analyzed to determine whether they produce mammalianhistamine H4 receptor protein. Identification of mammalian histamine H4receptor expressing host cell clones may be done by several means,including but not limited to immunological reactivity withanti-mammalian histamine H4 receptor antibodies, and the presence ofhost cell-associated mammalian histamine H4 receptor activity.

Expression of mammalian histamine H4 receptor DNA may also be performedusing in vitro produced synthetic mRNA. Synthetic mRNA or mRNA isolatedfrom mammalian histamine H4 receptor producing cells can be efficientlytranslated in various cell-free systems, including but not limited towheat germ extracts and reticulocyte extracts, as well as efficientlytranslated in cell based systems, including but not limited tomicroinjection into frog oocytes, with microinjection into frog oocytesbeing generally preferred.

To determine the mammalian histamine H4 receptor DNA sequence(s) thatyields optimal levels of mammalian histamine H4 receptor activity and/ormammalian histamine H4 receptor protein, human histamine H4 receptor DNAmolecules including, but not limited to, the following can beconstructed: the full-length open reading frame of the human histamineH4 receptor cDNA encoding the 44,495 Daltons protein from approximatelybase 1 to base 1173 (these numbers correspond to first nucleotide offirst methionine and last nucleotide before the first stop codon) andseveral constructs containing portions of the cDNA encoding humanhistamine H4 receptor protein. All constructs can be designed to containnone, all or portions of the 5′ or the 3′ untranslated region of humanhistamine H4 receptor cDNA. Human histamine H4 receptor activity andlevels of protein expression can be determined following theintroduction, both singly and in combination, of these constructs intoappropriate host cells. Following determination of the mammalianhistamine H4 receptor DNA cassette yielding optimal expression intransient assays, the mammalian histamine H4 receptor DNA construct istransferred to a variety of expression vectors, for expression in hostcells including, but not limited to, mammalian cells,baculovirus-infected insect cells, E. coli, and the yeast S. cerevisiae.

Host cell transfectants and microinjected oocytes may be used to assayboth the levels of mammalian histamine H4 receptor activity and levelsof mammalian histamine H4 receptor protein by the following methods. Inthe case of recombinant host cells, this involves the co-transfection ofone or possibly two or more plasmids, containing the mammalian histamineH4 receptor DNA encoding one or more fragments or subunits. In the caseof oocytes, this involves the co-injection of RNAs encoding mammalianhistamine H4 receptor protein. Following an appropriate period of timeto allow for expression, cellular protein is metabolically labelledwith, for example ³⁵S-methionine for 24 hours, after which cell lysatesand cell culture supernatants are harvested and subjected toimmunoprecipitation with polyclonal antibodies directed against themammalian histamine H4 receptor protein.

Other methods for detecting mammalian histamine H4 receptor activityinvolve the direct measurement of mammalian histamine H4 receptoractivity in whole cells transfected with mammalian histamine H4 receptorcDNA or oocytes injected with mammalian histamine H4 receptor mRNA.Mammalian histamine H4 receptor activity is measured by specific ligandbinding, for example [H³]-Histamine, and biological characteristics ofthe host cells expressing mammalian histamine H4 receptor DNA. In thecase of recombinant host cells and oocytes expressing mammalianhistamine H4 receptor cAMP quantitation and receptor binding techniquesare suitable examples of methods that can be used to measure mammalianhistamine H4 receptor activity and quantify mammalian histamine H4receptor protein.

Levels of mammalian histamine H4 receptor protein in host cells are alsoquantitated by immunoaffinity and/or ligand affinity techniques. Cellsexpressing mammalian histamine H4 receptor can be assayed for the numberof mammalian histamine H4 receptor molecules expressed by measuring theamount of radioactive histamine or histamine H4 ligand binding to cellmembranes. Mammalian histamine H4 receptor-specific affinity beads ormammalian histamine H4 receptor-specific antibodies are used to isolatefor example ³⁵S-methionine labelled or unlabelled mammalian histamine H4receptor protein. Labelled mammalian histamine H4 receptor protein isanalyzed by SDS-PAGE. Unlabelled mammalian histamine H4 receptor proteinis detected by Western blotting, ELISA or RIA assays employing mammalianhistamine H4 receptor specific antibodies.

Because the genetic code is degenerate, more than one codon may be usedto encode a particular amino acid, and therefore, the amino acidsequence can be encoded by any of a set of similar DNA oligonucleotides.Only one member of the set will be identical to the mammalian histamineH4 receptor sequence but will be capable of hybridizing to mammalianhistamine H4 receptor DNA even in the presence of DNA oligonucleotideswith mismatches under appropriate conditions. Under alternateconditions, the mismatched DNA oligonucleotides may still hybridize tothe mammalian histamine H4 receptor DNA to permit identification andisolation of mammalian histamine H4 receptor encoding DNA. Becausedifferent species have different codon usage preference, it ispreferable to prepare silent mutants of the mammalian Histamine H4receptor that contain optimized codon usage for the particularexpression host.

DNA encoding mammalian histamine H4 receptor from a particular organismmay be used to isolate and purify homologues of mammalian histamine H4receptor from other organisms. To accomplish this, the first mammalianhistamine H4 receptor DNA may be mixed with a sample containing DNAencoding homologues of mammalian histamine H4 receptor under appropriatehybridization conditions. The hybridized DNA complex may be isolated andthe DNA encoding the homologous DNA may be purified therefrom.

It is known that there is a substantial amount of redundancy in thevarious codons that code for specific amino acids. Therefore, thisinvention is also directed to those DNA sequences that containalternative codons that code for the eventual translation of theidentical amino acid. For purposes of this specification, a sequencebearing one or more replaced codons will be defined as a degeneratevariation. Also included within the scope of this invention aremutations either in the DNA sequence or the translated protein that doesnot substantially alter the ultimate physical properties of theexpressed protein. For example, substitution of aliphatic amino acidsalanine, valine, leucine and isoleucine, interchange of the hydroxylresidues serine and threonine, exchange of the acidic residues asparticacid and glutamic acid, substitution between the amide residuesasparagine and glutamine, exchange of the basic residues lysine andarginine and substitution among the aromatic residues phenylalanine,tyrosine may not cause a change in functionality of the polypeptide.Another example of a mutation that does not alter the functionalproperties of the receptor is construction of a chimeric gene expressinga different signal peptide that targets the receptor for translationwithin the endoplasmic reticulum. Such substitutions are well known andare described, for instance in Molecular Biology of the Gene, 4^(th) Ed.Bengamin Cummings Pub. Co. by Watson et al.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide having properties that are different than those ofthe naturally-occurring peptide. Methods of altering the DNA sequencesinclude, but are not limited to site directed mutagenesis or domainswapping (chimeric analysis). Chimeric genes are prepared by swappingdomains of similar or different genes to replace similar domains in themammalian histamine H4 receptor gene. Similarly, fusion genes may beprepared that add domains to the mammalian histamine H4 receptor gene,such as an affinity tag to facilitate identification and isolation ofthe gene. Fusion genes may be prepared to replace regions of themammalian histamine H4 receptor gene, for example to add a targetingsequence to redirect the normal transport of the protein or adding newpost-translational modification sequences to the mammalian histamine H4receptor gene (eg. a neoglycosylation site). Examples of alteredproperties include but are not limited to changes in the affinity of anenzyme for a substrate or a receptor for a ligand. All such changes ofthe polynucleotide or polypeptide sequences are anticipated as usefulvariants of the present invention so long as the original function ofthe polynucleotide or polypeptide sequence of the present invention ismaintained as described herein.

Identity or similarity, as known in the art, is relationships betweentwo or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. Both identityand similarity can be readily calculated (Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991). While there exists a numberof methods to measure identity and similarity between two polynucleotideor two polypeptide sequences, both terms are well known to skilledartisans (Sequence Analysis in Molecular Biology, von Heinje, G.,Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H.,and Lipman, D., (1988) SIAM J. Applied Math., 48, 1073. Methods commonlyemployed to determine identity or similarity between sequences include,but are not limited to those disclosed in Carillo, H., and Lipman, D.,(1988) SIAM J. Applied Math., 48, 1073. Preferred methods to determineidentity are designed to give the largest match between the sequencestested. Methods to determine identity and similarity are codified incomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, GCG program package (Devereux, J., et al., (1984) NucleicAcids Research 12(1), 387), BLASTP, BLASTN, and FASTA (Atschul, S. F. etal., (1990) J. Molec. Biol. 215, 403).

Polynucleotide(s) generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single- and double-stranded DNA, DNA that is a mixtureof single- and double-stranded regions or single-, double- andtriple-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded, or triple-stranded, or a mixture of single- anddouble-stranded regions. In addition, polynucleotide as used hereinrefers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The strands in such regions may be from the same molecule or fromdifferent molecules. The regions may include all of one or more of themolecules, but more typically involve only a region of some of themolecules. One of the molecules of a triple-helical region often is anoligonucleotide. As used herein, the term polynucleotide includes DNAsor RNAs as described above that contain one or more modified bases.Thus, DNAs or RNAs with backbones modified for stability or for otherreasons are “polynucleotides” as that term is intended herein. Moreover,DNAs or RNAs comprising unusual bases, such as inosine, or modifiedbases, such as tritylated bases, to name just two examples, arepolynucleotides as the term is used herein. It will be appreciated thata great variety of modifications have been made to DNA and RNA thatserve many useful purposes known to those of skill in the art. The termpolynucleotide as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including simple and complex cells, inter alia. Polynucleotidesembraces short polynucleotides often referred to as oligonucleotide(s).

The term polypeptides, as used herein, refers to the basic chemicalstructure of polypeptides that is well known and has been described intextbooks and other publications in the art. In this context, the termis used herein to refer to any peptide or protein comprising two or moreamino acids joined to each other in a linear chain by peptide bonds. Asused herein, the term refers to both short chains, which also commonlyare referred to in the art as peptides, oligopeptides and oligomers, forexample, and to longer chains, which generally are referred to in theart as proteins, of which there are many types. It will be appreciatedthat polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids,and that many amino acids, including the terminal amino acids, may bemodified in a given polypeptide, either by natural processes, such asprocessing and other post-translational modifications, but also bychemical modification techniques which are well known to the art. Eventhe common modifications that occur naturally in polypeptides are toonumerous to list exhaustively here, but they are well described in basictexts and in more detailed monographs, as well as in a voluminousresearch literature, and they are well known to those of skill in theart. Among the known modifications which may be present in polypeptidesof the present are, to name an illustrative few, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphatidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. Such modificationsare well known to those of skill and have been described in great detailin the scientific literature. Several particularly common modifications,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation, forinstance, are described in most basic texts, such as, for instancePROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,W.H. Freeman and Company, New York (1993). Many detailed reviews areavailable on this subject, such as, for example, those provided by Wold,F., Post-translational Protein Modifications: Perspectives andProspects, pgs. 1–12 in POSTRRANSLATIONAL COVALENT MODIFICATION OFPROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifteret al., (1990) Meth. Enzymol. 182, 626–646 and Rattan et al., “ProteinSynthesis: Posttranslational Modifications and Aging”, (1992) Ann. N.Y.Acad. Sci. 663, 48–62. It will be appreciated, as is well known and asnoted above, that polypeptides are not always entirely linear. Forinstance, polypeptides may be generally as a result of posttranslationalevents, including natural processing event and events brought about byhuman manipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationnatural process and by entirely synthetic methods, as well.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli or other cells, priorto proteolytic processing, almost invariably will be N-formylmethionine.During post-translational modification of the peptide, a methionineresidue at the amino terminus may be deleted. Accordingly, thisinvention contemplates the use of both the methionine-containing and themethionine-less amino terminal variants of the protein of the invention.The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as, forexample, E. coli. Accordingly, when glycosylation is desired, apolypeptide should be expressed in a glycosylating host, generally aeukaryotic cell. Insect cell often carry out the same posttranslationalglycosylations as mammalian cells and, for this reason, insect cellexpression systems have been developed to express efficiently mammalianproteins having native patterns of glycosylation, inter alia. Similarconsiderations apply to other modifications. It will be appreciated thatthe same type of modification may be present in the same or varyingdegree at several sites in a given polypeptide. Also, a givenpolypeptide may contain many types of modifications. In general, as usedherein, the term polypeptide encompasses all such modifications,particularly those that are present in polypeptides synthesizedrecombinantly by expressing a polynucleotide in a host cell.

Variant(s) of polynucleotides or polypeptides, as the term is usedherein, are polynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide, respectively. A variant of thepolynucleotide may be a naturally occurring variant such as a naturallyoccurring allelic variant, or it may be a variant that is not known tooccur naturally. (1) A polynucleotide that differs in nucleotidesequence from another, reference polynucleotide. Generally, differencesare limited so that the nucleotide sequences of the reference and thevariant are closely similar overall and, in many regions, identical. Asnoted below, changes in the nucleotide sequence of the variant may besilent. That is, they may not alter the amino acids encoded by thepolynucleotide. Where alterations are limited to silent changes of thistype a variant will encode a polypeptide with the same amino acidsequence as the reference. Also as noted below, changes in thenucleotide sequence of the variant may alter the amino acid sequence ofa polypeptide encoded by the reference polynucleotide. Such nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence, as discussed above. (2) A polypeptide that differs in aminoacid sequence from another, reference polypeptide. Generally,differences are limited so that the sequences of the reference and thevariant are closely similar overall and, in many regions, identical. Avariant and reference polypeptide may differ in amino acid sequence byone or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination. As used herein, a“functional derivative” of histamine H4 receptor is a compound thatpossesses a biological activity (either functional or structural) thatis substantially similar to the biological activity of histamine H4receptor. The term “functional derivatives” is intended to include the“fragments,” “variants,” “degenerate variants,” “analogs” and“homologues” or to “chemical derivatives” of histamine H4 receptor.Useful chemical derivatives of polypeptide are well known in the art andinclude, for example covalent modification of reactive organic sitecontained within the polypeptide with a secondary chemical moiety. Wellknown cross-linking reagents are useful to react to amino, carboxyl, oraldehyde residues to introduce, for example an affinity tag such asbiotin, a fluorescent dye, or to conjugate the polypeptide to a solidphase surface (for example to create an affinity resin). The term“fragment” is meant to refer to any polypeptide subset of histamine H4receptor. A molecule is “substantially similar” to histamine H4 receptorif both molecules have substantially similar structures or if bothmolecules possess similar biological activity. Therefore, if the twomolecules possess substantially similar activity, they are considered tobe variants even if the structure of one of the molecules is not foundin the other or even if the two amino acid sequences are not identical.The term “analog” refers to a molecule substantially similar in functionto either the entire histamine H4 receptor molecule or to a fragmentthereof. Further particularly preferred in this regard arepolynucleotides encoding variants, analogs, derivatives and fragments ofany one of SEQ.ID.NO.: 1, 5, 6, or 7, and variants, analogs andderivatives of the fragments, which have the amino acid sequencecorresponding to the polypeptide set forth in SEQ.ID.NO.:2, 8, 9, 10respectively in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 orno amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of the gene of any one of SEQ.ID.NO.: 1, 5, 6, or 7. Alsoespecially preferred in this regard are conservative substitutions. Mosthighly preferred are polynucleotides encoding polypeptides having theamino acid sequence of any one of SEQ.ID.NO.:2, 8, 9, and 10, withoutsubstitutions.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding the polypeptide having the amino acid sequence set out in anyone of SEQ.ID.NO.:2, 8, 9, 10, and polynucleotides which arecomplementary to such polynucleotides. Alternatively, highly preferredare polynucleotides that comprise a region that is at least 80%identical, more highly preferred are polynucleotides at comprise aregion that is at least 90% identical, and among these preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% identity are highly preferred amongthose with at least 95%, and among these those with at least 98% and atleast 99% are particularly highly preferred, with at least 99% being themost preferred. The polynucleotides which hybridize to thepolynucleotides of the present invention, in a preferred embodimentencode polypeptides which retain substantially the same biologicalfunction or activity as the polypeptide characterized by the deducedamino acid sequence of any one of SEQ.ID.NO.:2, 8, 9, or 10. Preferredembodiments in this respect, moreover, are polynucleotides that encodepolypeptides that retain substantially the same biological function oractivity as the mature polypeptide encoded by the DNA of any one ofSEQ.ID.NO.: 1, 5, 6, or 7. The present invention further relates topolynucleotides that hybridize to the herein above-described sequences.In this regard, the present invention especially relates topolynucleotides that hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term “stringentconditions” means hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences.

Polynucleotides of the present invention may be used as a hybridizationprobe for RNA, cDNA and genomic DNA to isolate full-length cDNAs andgenomic clones encoding the sequences of any one of SEQ.ID.NO.:1, 5, 6,or 7 and to isolate cDNA and genomic clones of other genes that have ahigh sequence similarity to any one of SEQ.ID.NO.:1, 5, 6, or 7. Suchprobes generally will comprise at least 15 bases. Preferably, suchprobes will have at least 30 bases and may have at least 50 bases.Particularly preferred probes will have at least 30 bases and will have50 bases or less. For example, the coding region of the gene of theinvention may be isolated by screening using the known DNA sequence tosynthesize an oligonucleotide probe. A labeled oligonucleotide having asequence complementary to that of a gene of the present invention isthen used to screen a library of cDNA, genomic DNA or mRNA to determineto which members of the library the probe hybridizes.

The polypeptides of the present invention include the polypeptide of anyone of SEQ.ID.NO.:2, 8, 9, 10 (in particular the mature polypeptide) aswell as polypeptides which have at least 70% identity to the polypeptideof any one of SEQ.ID.NO.:2, 8, 9, 10, preferably at least 80% identityto the polypeptide of any one of SEQ.ID.NO.:2, 8, 9, 10, and morepreferably at least 90% similarity (more preferably at least 90%identity) to the polypeptide of any one of SEQ.ID.NO.:2, 8, 9, 10 andstill more preferably at least 95% similarity (still more preferably atleast 97% identity) to the polypeptide of any one of SEQ.ID.NO.:2, 8, 9,10 and also include portions of such polypeptides with such portion ofthe polypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids. Representative examples ofpolypeptide fragments of the invention, include, for example, truncationpolypeptides of any one of SEQ.ID.NO.:2, 8, 9, 10. Truncationpolypeptides include polypeptides having the amino acid sequence of anyone of SEQ.ID.NO.:2, 8, 9, 10, or of variants or derivatives thereof,except for deletion of a continuous series of residues (that is, acontinuous region, part or portion) that includes the amino terminus, ora continuous series of residues that includes the carboxyl terminus or,as in double truncation mutants, deletion of two continuous series ofresidues, one including the amino terminus and one including thecarboxyl terminus. Also preferred in this aspect of the invention arefragments characterized by structural or functional attributes of thepolypeptide characterized by the sequences of any one of SEQ.ID.NO.:2,8, 9, 10. Preferred embodiments of the invention in this regard includefragments that comprise alpha-helix and alpha-helix forming regions,beta-sheet and beta-sheet-forming regions, turn and turn-formingregions, coil and coil-forming regions, hydrophilic regions, hydrophobicregions, alpha amphipathic regions, beta amphipathic regions, flexibleregions, surface-forming regions, substrate binding region, highantigenic index regions of the polypeptide of the invention, andcombinations of such fragments. Preferred regions are those that mediateactivities of the polypeptides of the invention. Most highly preferredin this regard are fragments that have a chemical, biological or otheractivity of the response regulator polypeptide of the invention,including those with a similar activity or an improved activity, or witha decreased undesirable activity.

Monospecific antibodies to mammalian histamine H4 receptor are purifiedfrom mammalian antisera containing antibodies reactive against mammalianhistamine H4 receptor or are prepared as monoclonal antibodies reactivewith mammalian histamine H4 receptor using the technique of Kohler andMilstein, Nature 256: 495–497 (1975). Monospecific antibody as usedherein is defined as a single antibody species or multiple antibodyspecies with homogenous binding characteristics for mammalian histamineH4 receptor. Homogenous binding as used herein refers to the ability ofthe antibody species to bind to a specific antigen or epitope, such asthose associated with the mammalian histamine H4 receptor, as describedabove. Mammalian histamine H4 receptor specific antibodies are raised byimmunizing animals such as mice, rats, guinea pigs, rabbits, goats,horses and the like, with rabbits being preferred, with an appropriateconcentration of mammalian histamine H4 receptor either with or withoutan immune adjuvant.

Preimmune serum is collected prior to the first immunization. Eachanimal receives between about 0.1 mg and about 1000 mg of mammalianhistamine H4 receptor associated with an acceptable immune adjuvant.Such acceptable adjuvants include, but are not limited to, Freund'scomplete. Freund's incomplete, alum-precipitate, water in oil emulsioncontaining Corynebacterium parvum and tRNA. The initial immunizationconsists of mammalian histamine H4 receptor in, preferably, Freund'scomplete adjuvant at multiple sites either subcutaneously (SC),intraperitoneally (IP) or both. Each animal is bled at regularintervals, preferably weekly, to determine antibody titer. The animalsmay or may not receive booster injections following the initialimmunization. Those animals receiving booster injections are generallygiven an equal amount of the antigen in Freund's incomplete adjuvant bythe same route. Booster injections are given at about three weekintervals until maximal titers are obtained. At about 7 days after eachbooster immunization or about weekly after a single immunization, theanimals are bled, the serum collected, and aliquots are stored at about−20° C.

Monoclonal antibodies (mAb) reactive with mammalian histamine H4receptor are prepared by immunizing inbred mice, preferably Balb/c, withmammalian histamine H4 receptor and any fragments thereof. The mice areimmunized by the IP or SC route with about 0.1 mg to about 10 mg,preferably about 1 mg, of mammalian histamine H4 receptor in about 0.5ml buffer or saline incorporated in an equal volume of an acceptableadjuvant, as discussed above. Freund's complete adjuvant is preferred.The mice receive an initial immunization on day 0 and are rested forabout 3 to about 30 weeks. Immunized mice are given one or more boosterimmunizations of about 0.1 to about 10 mg of mammalian histamine H4receptor in a buffer solution such as phosphate buffered saline by theintravenous (IV) route. Lymphocytes, from antibody positive mice,preferably splenic lymphocytes, are obtained by removing spleens fromimmunized mice by standard procedures known in the art. Hybridoma cellsare produced by mixing the splenic lymphocytes with an appropriatefusion partner, preferably myeloma cells, under conditions which willallow the formation of stable hybridomas. Fusion partners may include,but are not limited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 andSp 2/0, with Sp 2/0 being generally preferred. The antibody producingcells and myeloma cells are fused in polyethylene glycol, about 1000mol. wt., at concentrations from about 30% to about 50%. Fused hybridomacells are selected by growth in hypoxanthine, thymidine and aminopterinsupplemented Dulbecco's Modified Eagles Medium (DMEM) by proceduresknown in the art. Supernatant fluids are collected from growth positivewells on about days 14, 18, and 21 and are screened for antibodyproduction by an immunoassay such as solid phase immunoradioassay(SPIRA) using mammalian histamine H4 receptor as the antigen. Theculture fluids are also tested in the Ouchterlony precipitation assay todetermine the isotype of the mAb. Hybridoma cells from antibody positivewells are cloned by a technique such as the soft agar technique ofMacPhers on, Soft Agar Techniques, in Tissue Culture Methods andApplications, Kruse and Paterson, Eds., Academic Press, 1973.

Monoclonal antibodies are produced in vivo by injection of pristaneprimed Balb/c mice, approximately 0.5 ml per mouse, with about 2×10⁶ toabout 6×10⁶ hybridoma cells about 4 days after priming. Ascites fluid iscollected at approximately 8–12 days after cell transfer and themonoclonal antibodies are purified by techniques known in the art.

In vitro production of anti-mammalian histamine H4 receptor mAb iscarried out by growing the hybridoma in DMEM containing about 2% fetalcalf serum to obtain sufficient quantities of the specific mAb. The mAbare purified by techniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of mammalianhistamine H4 receptor in body fluids or tissue and cell extracts.

It is readily apparent to those skilled in the art that the abovedescribed methods for producing monospecific antibodies may be utilizedto produce antibodies specific for mammalian histamine H4 receptorpolypeptide fragments, or full-length nascent mammalian histamine H4receptor polypeptide, or the individual mammalian histamine H4 receptorsubunits. Specifically, it is readily apparent to those skilled in theart that monospecific antibodies may be generated which are specific foronly one mammalian histamine H4 receptor subunit or the fully functionalhistamine H4 receptor.

DNA clones, termed pH4R, are identified which encode proteins that, whenexpressed in any recombinant host, including but not limited tomammalian cells or insect cells or bacteria, form a mammalian histamineH4 receptor sensitive to histamine or other histamine H4 ligands. Theexpression of mammalian histamine H4 receptor DNA results in theexpression of the properties observed with mammalian histamine H4receptor.

Histamine is a biogenic amine transmitter that functions in somecapacity in nearly all physiological and pathophysiological situations.Histamine acts as a neurotransmitter and neuromodulator in the centralnervous system, mediates inflammatory and allergic responses, regulatesairway function, controls acid secretion in the stomach, regulatescardiovascular function as well as arterial and venous responses and iswithout doubt involved in processes yet to be determined. The histaminereceptors that mediate these effects are not completely characterized.One way to understand which histamine receptors are involved in theseprocesses is to develop chemical modulators (agonists, antagonists, andinverse agonists) of the receptors as research tools and therapeuticentities. Recombinant host cells expressing the mammalian histamine H4receptor can be used to provide materials for a screening method toidentify such agonists and antagonists. As such, this invention of themammalian histamine H4 receptor directly teaches a way to identify newagonists and antagonists that may prove useful as research tools or maybe used as therapeutics to treat disorders directly or indirectlyinvolving histamine receptors. Assays to detect compound interaction ormodulation of the histamine H4 receptor include, but are not limited to,direct ligand binding assays, competitive (or displacement) ligandbinding assays, or functional assays that measure the response of thereceptor to the ligand, for example by production of cAMP. Althoughthese assays are well known to those skilled in the art, they werepreviously no possible prior to obtaining the recombinant moleculestaught herein.

The present invention is also directed to methods for screening forcompounds that modulate the expression of DNA or RNA encoding mammalianhistamine H4 receptor as well as the function of mammalian histamine H4receptor protein in vivo. Compounds that modulate these activities maybe DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules.Compounds may modulate by increasing or attenuating the expression ofDNA or RNA encoding mammalian histamine H4 receptor, or the function ofmammalian histamine H4 receptor protein. Compounds that modulate theexpression of DNA or RNA encoding mammalian histamine H4 receptor or thefunction of mammalian histamine H4 receptor protein may be detected by avariety of assays. The assay may be a simple “yes/no” assay to determinewhether there is a change in expression or function. The assay may bemade quantitative by comparing the expression or function of a testsample with the levels of expression or function in a standard sample.Modulators identified in this process are useful as therapeutic agents,research tools, and diagnostic agents.

Kits containing mammalian histamine H4 receptor DNA or RNA, antibodiesto mammalian histamine H4 receptor, or mammalian histamine H4 receptorprotein may be prepared. Such kits are used to detect DNA thathybridizes to mammalian histamine H4 receptor DNA or to detect thepresence of mammalian histamine H4 receptor protein or peptide fragmentsin a sample. Such characterization is useful for a variety of purposesincluding but not limited to forensic analyses, diagnostic applications,and epidemiological studies.

The DNA molecules, RNA molecules, recombinant protein and antibodies ofthe present invention may be used to screen and measure levels ofmammalian histamine H4 receptor DNA, mammalian histamine H4 receptor RNAor mammalian histamine H4 receptor protein. The recombinant proteins,DNA molecules, RNA molecules and antibodies lend themselves to theformulation of kits suitable for the detection and typing of mammalianhistamine H4 receptor. Such a kit would comprise a compartmentalizedcarrier suitable to hold in close confinement at least one container.The carrier would further comprise reagents such as recombinantmammalian histamine H4 receptor protein or anti-mammalian histamine H4receptor antibodies suitable for detecting mammalian histamine H4receptor. The carrier may also contain a means for detection such aslabeled antigen or enzyme substrates or the like.

Nucleotide sequences that are complementary to the mammalian histamineH4 receptor encoding DNA sequence can be synthesized for antisensetherapy. These antisense molecules may be DNA, stable derivatives of DNAsuch as phosphorothioates or methylphosphonates, RNA, stable derivativesof RNA such as 2′-O-alkylRNA, or other mammalian histamine H4 receptorantisense oligonucleotide mimetics. Mammalian histamine H4 receptorantisense molecules may be introduced into cells by microinjection,liposome encapsulation or by expression from vectors harboring theantisense sequence, mammalian histamine H4 receptor antisense therapymay be particularly useful for the treatment of diseases where it isbeneficial to reduce mammalian histamine H4 receptor activity.

Mammalian histamine H4 receptor gene therapy may be used to introducemammalian histamine H4 receptor into the cells of target organisms. Themammalian histamine H4 receptor gene can be ligated into viral vectorswhich mediate transfer of the mammalian histamine H4 receptor DNA byinfection of recipient host cells. Suitable viral vectors includeretrovirus, adenovirus, adeno-associated virus, herpes virus, vacciniavirus, polio virus and the like. Alternatively, mammalian histamine H4receptor DNA can be transferred into cells for gene therapy by non-viraltechniques including receptor-mediated targeted DNA transfer usingligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofectionmembrane fusion or direct microinjection. These procedures andvariations thereof are suitable for ex vivo as well as in vivo mammalianhistamine H4 receptor gene therapy. Mammalian histamine H4 receptor genetherapy may be particularly useful for the treatment of diseases whereit is beneficial to elevate mammalian histamine H4 receptor activity.

Pharmaceutically useful compositions comprising mammalian histamine H4receptor DNA, mammalian histamine H4 receptor RNA, or mammalianhistamine H4 receptor protein, or modulators of mammalian histamine H4receptor activity, may be formulated according to known methods such asby the admixture of a pharmaceutically acceptable carrier. Examples ofsuch carriers and methods of formulation may be found in Remington'sPharmaceutical Sciences. To form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the protein, DNA, RNA, or modulator.

Therapeutic or diagnostic compositions of the invention are administeredto an individual in amounts sufficient to treat or diagnose disorders inwhich modulation of mammalian histamine H4 receptor-related activity isindicated. The effective amount may vary according to a variety offactors such as the individual's condition, weight, sex and age. Otherfactors include the mode of administration. The pharmaceuticalcompositions may be provided to the individual by a variety of routessuch as subcutaneous, topical, oral and intramuscular.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties that are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein may beused alone at appropriate dosages defined by routine testing in order toobtain optimal inhibition of the mammalian histamine H4 receptor or itsactivity while minimizing any potential toxicity. In addition,co-administration or sequential administration of other agents may bedesirable.

The present invention also has the objective of providing suitabletopical, oral, systemic and parenteral pharmaceutical formulations foruse in the novel methods of treatment of the present invention. Thecompositions containing compounds or modulators identified according tothis invention as the active ingredient for use in the modulation ofmammalian histamine H4 receptor receptors can be administered in a widevariety of therapeutic dosage forms in conventional vehicles foradministration. For example, the compounds or modulators can beadministered in such oral dosage forms as tablets, capsules (eachincluding timed release and sustained release formulations), pills,powders, granules, elixirs, tinctures, solutions, suspensions, syrupsand emulsions, or by injection. Likewise, they may also be administeredin intravenous (both bolus and infusion), intraperitoneal, subcutaneous,topical with or without occlusion, or intramuscular form, all usingforms well known to those of ordinary skill in the pharmaceutical arts.An effective but non-toxic amount of the compound desired can beemployed as a mammalian histamine H4 receptor modulating agent.

The daily dosage of the products may be varied over a wide range from0.01 to 1,000 mg per patient, per day. For oral administration, thecompositions are preferably provided in the form of scored or un-scoredtablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, and 50.0 milligrams of the active ingredient for the symptomaticadjustment of the dosage to the patient to be treated. An effectiveamount of the drug is ordinarily supplied at a dosage level of fromabout 0.0001 mg/kg to about 100 mg/kg of body weight per day. The rangeis more particularly from about 0.001 mg/kg to 10 mg/kg of body weightper day. The dosages of the mammalian histamine H4 receptor modulatorsare adjusted when combined to achieve desired effects. On the otherhand, dosages of these various agents may be independently optimized andcombined to achieve a synergistic result wherein the pathology isreduced more than it would be if either agent were used alone.

Advantageously, compounds or modulators of the present invention may beadministered in a single daily dose, or the total daily dosage may beadministered in divided doses of two, three or four times daily.Furthermore, compounds or modulators for the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, or via transdermal routes, using those forms of transdermalskin patches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing the compounds or modulators of the presentinvention is selected in accordance with a variety of factors includingtype, species, age, weight, sex and medical condition of the patient;the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

In the methods of the present invention, the compounds or modulatorsherein described in detail can form the active ingredient, and aretypically administered in admixture with suitable pharmaceuticaldiluents, excipients or carriers (collectively referred to herein as“carrier” materials) suitably selected with respect to the intended formof administration, that is, oral tablets, capsules, elixirs, syrups andthe like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitablyflavored suspending or dispersing agents such as the synthetic andnatural gums, for example, tragacanth, acacia, methyl-cellulose and thelike. Other dispersing agents which may be employed include glycerin andthe like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations which generally containsuitable preservatives are employed when intravenous administration isdesired.

Topical preparations containing the active drug component can be admixedwith a variety of carrier materials well known in the art, such as, eg.,alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils,mineral oil, PPG2 myristyl propionate, and the like, to form, eg.,alcoholic solutions, topical cleansers, cleansing creams, skin gels,skin lotions, and shampoos in cream or gel formulations.

The compounds or modulators of the present invention can also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The compounds or modulators of the presentinvention may also be coupled with soluble polymers as targetable drugcarriers. Such polymers can include polyvinyl-pyrrolidone, pyrancopolymer, polyhydroxypropylmethacryl-amidephenol,polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds ormodulators of the present invention may be coupled to a class ofbiodegradable polymers useful in achieving controlled release of a drug,for example, polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydro-pyrans,polycyanoacrylates and cross-linked or amphipathic block copolymers ofhydrogels.

For oral administration, the compounds or modulators may be administeredin capsule, tablet, or bolus form or alternatively they can be mixed inthe animals feed. The capsules, tablets, and boluses are comprised ofthe active ingredient in combination with an appropriate carrier vehiclesuch as starch, talc, magnesium stearate, or di-calcium phosphate. Theseunit dosage forms are prepared by intimately mixing the activeingredient with suitable finely-powdered inert ingredients includingdiluents, fillers, disintegrating agents, and/or binders such that auniform mixture is obtained. An inert ingredient is one that will notreact with the compounds or modulators and which is non-toxic to theanimal being treated. Suitable inert ingredients include starch,lactose, talc, magnesium stearate, vegetable gums and oils, and thelike. These formulations may contain a widely variable amount of theactive and inactive ingredients depending on numerous factors such asthe size and type of the animal species to be treated and the type andseverity of the infection. The active ingredient may also beadministered as an additive to the feed by simply mixing the compoundwith the feedstuff or by applying the compound to the surface of thefeed. Alternatively the active ingredient may be mixed with an inertcarrier and the resulting composition may then either be mixed with thefeed or fed directly to the animal. Suitable inert carriers include cornmeal, citrus meal, fermentation residues, soya grits, dried grains andthe like. The active ingredients are intimately mixed with these inertcarriers by grinding, stirring, milling, or tumbling such that the finalcomposition contains from 0.001 to 5% by weight of the activeingredient.

The compounds or modulators may alternatively be administeredparenterally via injection of a formulation consisting of the activeingredient dissolved in an inert liquid carrier. Injection may be eitherintramuscular, intra-ruminal, intratracheal, or subcutaneous. Theinjectable formulation consists of the active ingredient mixed with anappropriate inert liquid carrier. Acceptable liquid carriers include thevegetable oils such as peanut oil, cotton seed oil, sesame oil and thelike as well as organic solvents such as solketal, glycerol formal andthe like. As an alternative, aqueous parenteral formulations may also beused. The vegetable oils are the preferred liquid carriers. Theformulations are prepared by dissolving or suspending the activeingredient in the liquid carrier such that the final formulationcontains from 0.005 to 10% by weight of the active ingredient.

Topical application of the compounds or modulators is possible throughthe use of a liquid drench or a shampoo containing the instant compoundsor modulators as an aqueous solution or suspension. These formulationsgenerally contain a suspending agent such as bentonite and normally willalso contain an antifoaming agent. Formulations containing from 0.005 to10% by weight of the active ingredient are acceptable. Preferredformulations are those containing from 0.01 to 5% by weight of theinstant compounds or modulators.

The following examples illustrate the present invention without,however, limiting the same thereto.

EXAMPLE 1

Cloning of Human Histamine H4 Receptor DNA (pH4R)

cDNA synthesis:

First strand synthesis: Approximately 5 μg of human bone marrow mRNA(Clonetech) was used to synthesize cDNA using the cDNA synthesis kit(Life Technologies). 2 μl of Not1 primmer adapter was added to 5 μl ofmRNA and the mixture was heated to 70° C. for 10 minutes and placed onice. The following reagents were added on ice: 4 μl of 5× first strandbuffer (250 mM TRIS-HCl (pH8.3), 375 mM KCl, 15 mMMgCl₂), 2 μl of 0.1MDTT, 10 mM dNTP (nucleotide triphosphates) mix and 1 μl of DEPC treatedwater. The reaction was incubated at 42° C. for 5 minutes. Finally, 5 μlof Superscript RT II was added and incubated at 42° C. for 2 more hours.The reaction was terminated on ice.Second strand synthesis: The first strand product was adjusted to 93 μlwith water and the following reagents were added on ice:30 μl of 5× 2ndstrand buffer (100 mM TRIS-HCl (pH6.9), 450 mM KCl, 23 mM MgCl₂, 0.75 mMβ-AND+, 50 mM (NH₄)₂SO₄), 3 μl of 10 mM dNTP (nucleotide triphosphates),1 μl E. coli DNA ligase (10 units) 1 μl RNase H (2 units), 4 μl DNA polI (10 units). The reaction was incubated at 16° C. for 2 hours. The DNAfrom second strand synthesis was treated with T4 DNA polymerase andplaced at 16° C. to blunt the DNA ends. The double stranded cDNA wasextracted with 150 μl of a mixture of phenol and chloroform (1:1, v:v)and precipitated with 0.5 volumes of 7.5 M NH4OAc and 2 volumes ofabsolute ethanol. The pellet was washed with 70% ethanol and dried downat 37° C. to remove the residual ethanol. The double stranded DNA pelletwas resuspended in 25 μl of water and the following reagents were added;10 μl of 5× T4 DNA ligase buffer, 10 μl of Sal1 adapters and 5 μl of T4DNA ligase. The ingredients were mixed gently and ligated overnight at16° C. The ligation mix was extracted with phenol:chloroform:isoamylalcohol, vortexed thoroughly and centrifuged at room temperature for 5minutes at 14,000×g to separate the phases. The aqueous phase wastransferred to a new tube and the volume adjusted to 100 ml with water.The purified DNA was size selected on a chromaspin 1000 column(Clontech) to eliminate the smaller cDNA molecules. The double strandedDNA was digested with Not1 restriction enzyme for 34 hours at 37° C. Therestriction digest was electrophoresed on a 0.8% low melt agarose gel.The cDNA in the range of 1–5 kb was cut out and purified using Gelzyme(Invitrogen). The product was extracted with phenol:chloroform andprecipitated with NH₄OAc and absolute ethanol. The pellet was washedwith 70% ethanol and resuspended in 10 ml of water.Ligation of cDNA to the Vector The cDNA was split up into 5 tubes (2 μleach) and the ligation reactions were set up by adding 4.5 μl of water,2 μl of 5× ligation buffer, 1 μl of p-Sport vector DNA (cut withSal-1/Not1 and phosphatase treated) and 0.5 μl of T4 DNA ligase. Theligation was incubated at 40° C. overnight.Introduction of Ligated cDNA into E. coli by Electroporation:The ligation reaction volume was adjusted to a total volume of 20 μlwith water. Five ml of yeast tRNA, 12.5 ml of 7.5M NH₄OAc and 70 ml ofabsolute ethanol (−20° C.) was added. The mixture was vortexedthoroughly, and immediately centrifuged at room temperature for 20minutes at 14,000× g. The pellets were washed in 70% ethanol and eachpellet was resuspended in 5 ml of water. All 5 ligations (25 ml) werepooled and 100 μl of DH10B electro-competent cells (Life Technologies)were electroporated with 1 μl of DNA (total of 20 electroporations),then plated out on ampicillin plates to determine the number ofrecombinants (cfu) per μl. The entire library was seeded into 2 litersof Super Broth and maxipreps were made using Promega Maxi Prep kit andpurified on cesium chloride gradients.Screening of Library:

1 μl aliquots of the library constructed above were electroporated intoElectromax DH10B cells (Life Technologies). The volume was adjusted to 1ml with SOC media and incubated for 1 hour at 37° C. with shaking. Thelibrary was then plated out on 50 150 cm² plates containing LB to adensity of 5000 colonies per plate. These were grown overnight at 37° C.

A histamine H4 receptor probe was generated by polymerase chain reactionusing the following primer pair. 5′ oligo: 5′ACTAGAATTCACCGTGATGCCAGATACTAATAGCACA 3′ [SEQ.ID.NO.:1] and 3′ oligo: 5′ATGCAGGATCCAGCATTTGAGACTGACAGGTAT 3′ [SEQ.ID.NO.:2]. Amplification wascycled 35 times with a 50–60° C. annealing temperature and humanthalamus cDNA as template. The PCR fragment that was generated (400–500bp) was 32P-labelled using the klenow fragment of DNA polymerase I andan oligo-labeling kit (Pharmacia). The fragment was then cleaned by onepassage through a S-200 column (Pharmacia).

The library colonies are lifted on nitrocellulose filters andcross-linked via UV irradiation (Stratagene). Filters were washed threetimes in buffer (50 mM TRIS, 1 M NaCl, 2 mM EDTA, 1% SDS) at 42° C.Filters were then pre-hybridized in 1:1 Southern Prehyb:Formamide withsalmon sperm DNA (50 mg, boiled) for 6 hours at 42° C. Filters were thenhybridized with the probe (1×10⁶ counts/ml) overnight The filters werethen washed one time with 2×SSC/0.2% SDS at room temperature for 15minutes, 2 times with 0.2×SSC/0.1% SDS at 45° C. for 30 minutes each.Filters were then wrapped in plastic wrap and exposed to film (Kodak)overnight at −80° C.

Positive clones were identified. Resulting positives were cored from theoriginal plate, incubated in LB for 45 minutes at 37° C. and re-platedovernight. The filter lifting/hybridizing/washing/colony pickingprocedure was replicated until a single clone or clones were isolated,representing an individual cDNA.

From the screen for human histamine H4 receptor, all cDNA clones wereisolated and sequenced. One clone, pH4R, contained a 1173 bp insert(FIG. 1). This sequence had an apparent open reading frame fromnucleotide 1 to 1173. This open reading frame encoded a protein of 371amino acids (FIG. 2).

EXAMPLE 2

Cloning of Human Histamine H4 Receptor cDNA into a Mammalian ExpressionVector

The human histamine H4 receptor cDNAs (collectively referred to as pH4R)were cloned into the mammalian expression vector pCIneo. The humanhistamine H4 receptor cDNA clone was isolated from the human thalamuscDNA library. The full length cDNA was used as the template for PCRusing specific primers with EcoR1 (5′ACT AGA ATT CGC CAC CAT GCC AGA TACTAA TAG CACA3′) [SEQ.ID.NO.:3] and Not1 (5′ACT ACT GCG GCC GCT TAA GAAGAT ACT GAC CGA CTGT3′) [SEQ.ID.NO.:4] sites for cloning. The PCRproduct was purified on a column (Wizard PCR DNA purification kit fromPromega) and digested with Not I and EcoR1 (NEB) to create cohesiveends. The product was purified by a low melting agarose gelelectrophoresis. The pCIneo vector was digested with EcoR1 and Not1enzymes and subsequently purified on a low melt agarose gel. The linearvector was used to ligate to the human histamine H4 receptor cDNAinserts. Recombinants were isolated, designated human histamine H4receptor, and used to transfect mammalian cells (SK-N-MC cells) byCaPO₄-DNA precipitation. Stable cell clones were selected by growth inthe presence of G418. Single G418 resistant clones were isolated andshown to contain the intact human histamine H4 receptor gene. Clonescontaining the human histamine H4 receptor cDNAs were analyzed for pH4Rexpression by measuring inhibition of adenylate cyclase in response tohistamine according to the method of (Konig, Mahan et al. 1991) or bydirectly measuring cAMP accumulation by radioimmunoassay usingFlashplates (NEN). Expression was also analyzed using [³H]-histaminebinding assays (Clark, Korte et al. 1992). Recombinant plasmidscontaining human histamine H4 receptor encoding DNA were used totransform the mammalian COS7 or CHO cells or HEK293 or L-cells orSK-N-MC cells.

Cells expressing human histamine H4 receptor, stably or transiently, areused to test for expression of human histamine H4 receptor and for[³H]-histamine binding activity (FIG. 4). These cells are used toidentify and examine other compounds for their ability to modulate,inhibit or activate the human histamine H4 receptor and to compete forradioactive histamine binding.

Cassettes containing the human histamine H4 receptor cDNA in thepositive orientation with respect to the promoter are ligated intoappropriate restriction sites 3′ of the promoter and identified byrestriction site mapping and/or sequencing. These cDNA expressionvectors are introduced into fibroblastic host cells for example COS-7(ATCC# CRL1651), and CV-1 tat [Sackevitz et al., Science 238: 1575(1987)], 293, L (ATCC# CRL6362), SK-N-MC (ATCC# HTB-10) by standardmethods including but not limited to electroporation, or chemicalprocedures (cationic liposomes, DEAE dextran, calcium phosphate).Transfected cells and cell culture supernatants are harvested andanalyzed for human histamine H4 receptor expression as described herein.

All of the vectors used for mammalian transient expression can be usedto establish stable cell lines expressing human histamine H4 receptor.Unaltered human histamine H4 receptor cDNA constructs cloned intoexpression vectors are expected to program host cells to make humanhistamine H4 receptor protein. The transfection host cells include, butare not limited to, CV-1-P [Sackevitz et al., Science 238: 1575 (1987)],tk-L [Wigler, et al. Cell 11: 223 (1977)], NS/0, and dHFr-CHO [Kaufmanand Sharp, J. Mol. Biol. 159: 601, (1982)].

Co-transfection of any vector containing human histamine H4 receptorcDNA with a drug selection plasmid including, but not limited to G418,aminoglycoside phosphotransferase; hygromycin, hygromycin-Bphosphotransferase; APRT, xanthine-guanine phosphoribosyl-transferase,will allow for the selection of stably transfected clones. Levels ofhuman histamine H4 receptor are quantitated by the assays describedherein.

Human histamine H4 receptor cDNA constructs are also ligated intovectors containing amplifyable drug-resistance markers for theproduction of mammalian cell clones synthesizing the highest possiblelevels of human histamine H4 receptor. Following introduction of theseconstructs into cells, clones containing the plasmid are selected withthe appropriate agent, and isolation of an over-expressing clone with ahigh copy number of plasmids is accomplished by selection in increasingdoses of the agent.

The expression of recombinant human histamine H4 receptor is achieved bytransfection of full-length human histamine H4 receptor cDNA into amammalian host cell.

Characterization of Human Histamine H4 Receptor

Human SK-N-MC cells were transfected with pH4R and selected in thepresence of neomycin for 10 days. Individual colonies were picked andgrown in 6-well dishes. Cells were then plated onto 96-well plates andgrown to confluence. Cells were incubated for 20 minutes withisobutylmethylxanthine (1 mM). Cells were then stimulated with histamine(100 pM–100 uM) for 5 minutes. Cells were then stimulated with forskolin(3 uM) and allowed to incubate at 37° C. for 20 minutes. Cells were thentreated with 0.1N hydrochloric acid. Cells were then frozen and thawed.Aliquots of the supernatant were then analyzed for their cyclic AMPcontent using a standard cAMP radioimmunoassay kit (Flashplates, NEN).The forskolin treatment raises the intracellular concentration of cAMP.Any cells that responded to histamine by decreasing the cAMP content inresponse to forskolin were considered to be expressing active functionalhuman histamine H4 receptor. The recombinant human histamine H4 receptorexpressed from the human histamine H4 receptor-encoding DNA moleculedescribed herein was shown to be specifically activated by histamine.

EXAMPLE 3 Binding Assay on Recombinant Human Histamine H4 Receptor

SK-N-MC cells or COS7 cells that were transiently transfected with pH4Rand grown in 150 cm2 tissue culture dishes. Cells were washed withsaline solution, scraped with a cell scraper and collected bycentrifugation (1000 rpm, 5 min). SK-N-MC or COS7 cells expressing humanhistamine H4 receptor binds ³H-histamine with high affinity (FIG. 4).Cell membranes are prepared by homogenization of the cell pellet in 20mM Tris-HCl with a polytron tissue homogenizer for 10 seconds at highspeed. Homogenate is centrifuged at 1000 rpm for 5 minutes at 4° C. Thesupernatant is then collected and centrifuged at 20,000×g for 25 minutesat 4° C. The final pellet is resuspended in 50 mM Tris-HCl. Cellmembranes are incubated with ³H-histamine (0.5 nM–70 nM) in the presenceor absence of excess histamine (10000 nM). Incubation occurs at roomtemperature for 45 minutes. Membranes are harvested by rapid filtrationover Whatman GF/C filters and washed 4 times with ice cold 50 mM TrisHCl. Filters are then dried, mixed with scintillant and counted forradioactivity. SK-N-MC or COS7 cells expressing human histamine H4receptor are used to measure the affinity of binding of other compoundsand their ability to displace ³H-ligand binding by incubating the abovedescribed reaction in the presence of various concentrations ofinhibitor or compound to be tested.

EXAMPLE 4

Primary Structure Of The human histamine H4 receptor Protein

The nucleotide sequences of pH4R receptor revealed a single large openreading frame of about 1173 base pairs. The first in-frame methioninewas designated as the initiation codon for an open reading frame thatpredicts a human histamine H4 receptor protein with an estimatedmolecular mass (M_(r)) of about 44495.

The predicted human histamine H4 receptor protein was aligned withnucleotide and protein databases and found to be related to the humanhistamine H1, human histamine H2 receptors, and human histamine H3receptors. Approximately 25% of the amino acids in human histamine H4receptor were highly conserved, showing at least 25% amino acid identitywithin the histamine H2 receptor, 28% with the histamine H1 receptor,38% with the human H3 receptor, and approximately 25% with the family ofbiogenic amine G-protein coupled receptors. The conserved motifs foundin this family of receptors, such as seven conserved hydrophobicdomains, were also found in the human histamine H4 receptor sequence.The human histamine H4 receptor protein contained the conservedaspartate residue found in the 3^(rd) transmembrane domain of allbiogenic amine receptors. The human histamine H4 receptor proteincontained the conserved asparagine residue found in the 1^(st)transmembrane domain of all biogenic amine receptors. The humanhistamine H4 receptor protein contained the conserved arginine residuefound in the 3^(rd) transmembrane domain of all biogenic aminereceptors. The human histamine H4 receptor protein contained theconserved tryptophan residue found in the 4^(th) transmembrane domain ofall biogenic amine receptors. The human histamine H4 receptor proteincontained the conserved phenylalanine residue found in the 5^(th)transmembrane domain of all biogenic amine receptors. The humanhistamine H4 receptor protein contained the conserved proline residuefound in the 6^(th) transmembrane domain of all biogenic aminereceptors. The human histamine H4 receptor protein contained theconserved tyrosine residue found in the 7^(th) transmembrane domain ofall biogenic amine receptors.

EXAMPLE 5

Cloning of the Human Histamine H4 Receptor cDNA into E. coli ExpressionVectors

Recombinant human histamine H4 receptor is produced in E. coli followingthe transfer of the human histamine H4 receptor expression cassette intoE. coli expression vectors, including but not limited to, the pET series(Novagen). The pET vectors place human histamine H4 receptor expressionunder control of the tightly regulated bacteriophage T7 promoter.Following transfer of this construct into an E. coli host which containsa chromosomal copy of the T7 RNA polymerase gene driven by the induciblelac promoter, expression of human histamine H4 receptor is induced whenan appropriate lac substrate (IPTG) is added to the culture. The levelsof expressed human histamine H4 receptor are determined by the assaysdescribed herein.

The cDNA encoding the entire open reading frame for human histamine H4receptor is inserted into the NdeI site of pET [16] 11a. Constructs inthe positive orientation are identified by sequence analysis and used totransform the expression host strain BL21. Transformants are then usedto inoculate cultures for the production of human histamine H4 receptorprotein. Cultures may be grown in M9 or ZB media, whose formulation isknown to those skilled in the art. After growth to an OD₆₀₀=1.5,expression of human histamine H4 receptor is induced with 1 mM IPTG for3 hours at 37° C.

EXAMPLE 6 Cloning of Human Histamine H4 Receptor cDNA into a BaculovirusExpression Vector for Expression in Insect Cells

Baculovirus vectors, which are derived from the genome of the AcNPVvirus, are designed to provide high level expression of cDNA in the Sf9line of insect cells (ATCC CRL# 1711). Recombinant baculovirusexpressing human histamine H4 receptor cDNA is produced by the followingstandard methods (In Vitrogen Maxbac Manual): the human histamine H4receptor cDNA constructs are ligated into the polyhedrin gene in avariety of baculovirus transfer vectors, including the pAC360 and theBlueBac vector (InVitrogen). Recombinant baculovirus are generated byhomologous recombination following co-transfection of the baculovirustransfer vector and linearized AcNPV genomic DNA [Kitts, P. A., Nuc.Acid. Res. 18: 5667 (1990)] into Sf9 cells. Recombinant pAC360 virusesare identified by the absence of inclusion bodies in infected cells andrecombinant pBlueBac viruses are identified on the basis ofβ-galactosidase expression (Summers, M. D. and Smith, G. E., TexasAgriculture Exp. Station Bulletin No. 1555). Following plaquepurification, human histamine H4 receptor expression is measured by theassays described herein.

The cDNA encoding the entire open reading frame for human histamine H4receptor is inserted into the BamHI site of pBlueBacII. Constructs inthe positive orientation are identified by sequence analysis and used totransfect Sf9 cells in the presence of linear AcNPV mild type DNA.

Authentic, active human histamine H4 receptor is found in the cytoplasmof infected cells. Active human histamine H4 receptor is extracted frominfected cells by hypotonic or detergent lysis.

EXAMPLE 7 Cloning of Human Histamine H4 Receptor cDNA into a YeastExpression Vector

Recombinant human histamine H4 receptor is produced in the yeast S.cerevisiae following the insertion of the optimal human histamine H4receptor cDNA cistron into expression vectors designed to direct theintracellular or extracellular expression of heterologous proteins. Inthe case of intracellular expression, vectors such as EmBLyex4 or thelike are ligated to the human histamine H4 receptor cistron [Rinas, U.et al., Biotechnology 8: 543–545 (1990); Horowitz B. et al., J. Biol.Chem. 265: 4189–4192 (1989)]. For extracellular expression, the humanhistamine H4 receptor cistron is ligated into yeast expression vectorswhich fuse a secretion signal (a yeast or mammalian peptide) to the NH₂terminus of the human histamine H4 receptor protein [Jacobson, M. A.,Gene 85: 511–516 (1989); Riett L. and Bellon N. Biochem. 28: 2941–2949(1989)).

These vectors include, but are not limited to pAVE1>6, which fuses thehuman serum albumin signal to the expressed cDNA (Steep O. Biotechnology8: 42–46 (1990)], and the vector pL8PL which fuses the human lysozymesignal to the expressed cDNA [Yamamoto, Y., Biochem. 28: 2728–2732)]. Inaddition, human histamine H4 receptor is expressed in yeast as a fusionprotein conjugated to ubiquitin utilizing the vector pVEP [Ecker, D. J.,J. Biol. Chem. 264: 7715–7719 (1989), Sabin, E. A., Biotechnology 7:705–709 (1989), McDonnell D. P., Mol. Cell Biol. 9: 5517–5523 (1989)].The levels of expressed human histamine H4 receptor are determined bythe assays described herein.

EXAMPLE 8 Cloning Murine, Rat, and Guinea Pig Histamine H4 ReceptorcDNAs

Two primers, forward primer: 5′GTG GTG GAC AAA AAC CTT AGA CAT CGA AGT3′[SEQ.ID.NO.:11], and reverse primer: 5′ACT GAG ATG ATC ACG CTT CCA CAGGCT CCA3′ [SEQ.ID.NO.:12] were used to amplify a 500 bp cDNA fragmentfrom mouse spleen, rat spleen, and guinea pig bone marrow cDNAlibraries. These cDNA fragments were sub-cloned into the pPCR2 vector(Invitrogen). The resulting DNA sequence showed 65–70% identity to humanH4 sequence and this region of each clone was used as anchoring regionsto clone the 5′ and 3′ ends by RACE methodology.Cloning of Mouse H₄Full Length cDNAThe mouse H4 cDNA 5′end was PCR amplified by the rapid amplification ofcDNA end (RACE) method from mouse spleen Marathron-Ready cDNA (Clontech)using the adaptor primer (AP): 5′ CCA TCC TAA TAC GAC TCA CTA TAG GGC 3′[SEQ.ID.NO.:13] and mouse gene specific primer P1: 5′ CAC TCT GTA ACAAAG CCA GGC TCA CAG TC 3′ [SEQ.ID.NO.:14]. The mouse H4 cDNA 3′ end wasRACE amplified from mouse spleen Marathron-Ready cDNA (Clontech) usingthe AP and mouse H4 specific gene primer P2: 5′ TGC ATC TCG TCA CTC AGAAAG TCC TCG AAG 3′ [SEQ.ID.NO.:15]. The 5′ end and 3′ end RACE productsof mouse histamine H4 receptor cDNA were sequenced and the complete cDNAsequence assembled. The coding region of mouse H4 was then PCR amplifiedfrom mouse spleen cDNA using two primers, forward primer 5′ ACG AGA ATTCGC CAC CAT GTC GGA GTC TAA CAG TAC TGG 3′ [SEQ.ID.NO.:16] and reverseprimer: 5′ ATG ACA GCG GCC GCA GTT GGC ACT CGT GAA GCA ATT CTC 3′[SEQ.ID.NO.:17]. The full-length cDNA PCR product was cloned into themammalian expression vector pCINeo (Promega).Cloning of rat H4 cDNA.Similar to that of mouse, the rat histamine H4 receptor cDNA 5′ and 3′ends were cloned by RACE from a rat spleen cDNA library (Marathon-ReadycDNA-Clonetech) using P3: 5′ CAT TGG GCC ATT GAC CAA GAA AGC CAG TAT C3′[SEQ.ID.NO.:18] and P4: 5′ TCA TTC AGA AAG TCC ACG AGG AAA GAG CAG 3′[SEQ.ID.:19] together with the primer AP, described supra. The RACE cDNAproduct was sequenced and the complete cDNA sequence assembled. Thecoding region of rat histamine H4 receptor was PCR amplified from therat spleen cDNA library using two primers, forward primer 5′ ACG TGA ATTCGC CAC CAT GTC GGA GTC TAA CGG CAC TGA 3′ [SEQ.ID.NO.:20] and reverseprimer. 5′ ACT GAT GCG GCC GCG AAG CTG GCA CAC ATG AAG CTT CTC 3′[SEQ.ID.NO.:21]. The full-length cDNA product was cloned into themammalian expression vector pCINeo.Cloning the Guinea Pig H4 cDNA Full Length

Guinea pig bone marrow RNA was purified using a RNA purification kitTrizol (Gibco-BRL) and cDNA first strand was synthesized using the smartcDNA synthesis system (Clontech) per the manufacture's instructions.This cDNA library was used to clone the guinea pig histamine H4 receptor5′ end and 3′ end by RACE methodology using guinea pig H4 specificprimers P5: 5′ ATA ATG ATG TAG GGA GAG CAA AGT ACC ACT 3′[SEQ.ID.NO.:22] and P6: 5′ ACA CTC CTG CAG ACA GGA CCC CGA TTC AAG 3′[SEQ.ID.NO.:23] together with the adaptor primer provided by themanufacturer. The race products were sequenced and the complete cDNAsequence assembled. The complete coding region of guinea pig histamineH4 receptor was then PCR amplified from the bone marrow cDNA pool usingtwo primers: forward primer 5′ ACG TCT CGA GGC CAC CAT GTT GGC AAA TAACAG TAC AAT CG 3′ [SEQ.ID.NO.:24] and reverse primer: 5′ ACG ACA GCG GCCGCC TTC AAG TGG ATA TTG AGC GGT TGT GT 3′ [SEQ.ID.NO.:25]. Thefull-length cDNA clone was cloned into the mammalian expression vectorpCINeo.

The complete polynucleotide coding sequence for murine, rat, and guineapig are shown in FIG. 5. The corresponding amino acid sequences areshown in FIG. 6, the alignment of human, murine, rat, and guinea pigpolynucleotides are shown in FIG. 7, and the alignment of human, murine,rat, and guinea pig polypeptides are shown in FIG. 8. The percenthomology between the human, rat, guinea pig and mouse nucleotidesequences was determined using Vector NTI Suite 6.0 (Informatix, Inc.),and the results are shown in Table 1.

TABLE 1 Human Murine Rat Guinea Pig Human 100 72.8 72.5 75.6 Murine 10088.4 75.3 Rat 100 74.5 Guinea Pig 100

EXAMPLE 9 Ligand Binding to Mammalian Histamine H4 Receptors.

The affinity of ³H-histamine for rat, mouse, guinea pig, and humanhistamine H4 receptors was determined using standard techniques asdescribed herein. Saturation binding was performed on membranes fromSK-N-MC cells stably transfected with the appropriate histamine H4receptor. The Kd values were derived from a −1/slope of the linearregression of a Scatchard plot (bound/free vs bound). The results areshow in Table 2.

TABLE 2 ³H-histamine K_(d) Species (nM) Rat 105 Murine 34 Guinea Pig 20Human 5The relative affinity of several known histamine receptor ligands wasdetermined by competitive binding of 30 nM ³H-histamine. K_(i) valuesfor each ligand were calculated according to the method of Cheng andPruscoff (K_(i)≈IC₅₀/(1+[³H-histamine]/K_(d)). The Kd values for³H-histamine were those set forth in Table 2. The results are presentedin Table 3.

TABLE 3 Human Guinea Pig Rat Murine Compound Ki (nM) Ki (nM) Ki (nM) Ki(nM) Imetit 1.3 30 6.8 6.6 Histamine 5.9 27 70 41 Clobenpropit 4.9 3.663 14 N-methylhistamine 48 220 552 303 Thioperamide 52 83 28 22R-α-methylhistamine 144 486 698 382 Burimamide 124 840 958 696 Clozapine626 185 2200 2780

EXAMPLE 10

RT-PCR Detection of Human H4 mRNA Expression.

PCR primers were used to expand a human Histamine H4 receptor cDNAfragment in cDNA libraries of cerebellum, cortex, hypothalamus, smallintestine, dorsal root ganglia (DRG), hippocampus, spleen, thalamus,placenta, heart, liver, lung, uterus, pituitary, spinal cord, and bonemarrow under condition of 94 C 45 sec, 60 C 45 sec, 72 C 2 min for 35cycles. The PCR products were run on a 1% agarose gel and DNA wasstained with ethidium bromide (10 ug/ml) and visualized with UV. The PCRproducts in gel were then transferred to a nitrocellulose membrane andhybridized with a ³²P-labeled human H4 DNA probe. As seen in FIG. 3, thehuman Histamine H4 receptor is highly expressed in the bone marrow.Similar experiments were conducted for mouse, rat, and guinea pighistamine H4 receptor. In all species, the histamine H4 receptor ishighly expressed in the bone marrow.

REFERENCES

-   Arrang, J. M., M. Garbarg, et al. (1983). “Autoinhibition of brain    histamine release mediated by a novel class (H3) of histamine    receptor.” Nature (London) 302(5911): 832–7.-   Clark, M. A., A. Korte, et al. (1993). “Guanine nucleotides and    pertussis toxin reduce the affinity of histamine H3 receptors on    AtT-20 cells.” Agents Actions 40(3–4): 129–34.-   Clark, M. A., A. Korte, et al. (1992). “High affinity histamine H3    receptors regulate ACTH release by AtT-20 cells.” Eur. J. Pharmacol.    210(1): 31–5.-   De Vos, C. (1999). “H1-receptor antagonists: Effects on leukocytes,    myth or reality.” Clin. Exp. Allergy 29(Suppl.3):60–63-   Gantz, I., M. Schaffer, et al. (1991). “Molecular cloning of a gene    encoding the histamine H2 receptor.” Proc. Natl. Acad. Sci. U.S.A.    88(2): 429–33.-   Hill, S. J., C. R. Ganellin, et al. (1997). “International Union of    Pharmacology. XIII. Classification of histamine receptors.”    Pharmacol. Rev. 49(3): 253–278.-   Konig, M., L C. Mahan, et al. (1991). “Method for identifying    ligands that bind to cloned Gs- or Gi-coupled receptors.” Mol. Cell.    Neurosci. 2(4): 331–7.-   Lovenberg, T. W., B. L. Roland, et al. (1999) “Cloning and    functional expression of the human histamine H3 receptor.” Mol.    Pharmacology 55:1101–1107.-   Pollard. H., J. Moreau, et al. (1993). “A detailed autoradiographic    mapping of histamine H3 receptors in rat brain areas.” Neuroscience    (Oxford) 52(1): 169–89.-   Raible, D. G., Lenahan, T., et al. (1994) “Pharmacologic    characterization of a novel histamine receptor on human    eosinophils.” Am. J. Respir. Crit. Care Med. 149:1506–1511-   Yamashita, M., H. Fukui, et al. (1991). “Expression cloning of a    cDNA encoding the bovine histamine H1 receptor.” Proc. Natl. Acad.    Sci. U.S.A. 88(24): 11515–19.

1. An isolated and purified nucleic acid molecule that encodes a murinehistamine H4 receptor protein, or a complement of said nucleic acidmolecule, comprising a member selected from the group consisting of: (a)a polynucleotide sequence encoding a polypeptide comprising amino acids1 to 391 of SEQ ID NO:8; and (b) a polynucleotide sequence which is afull-length complement of a polynucleotide sequence encoding amino acids1 to 391 of SEQ ID NO:8.
 2. The nucleic acid molecule of claim 1 whereinthe polynucleotide is RNA.
 3. The nucleic acid molecule of claim 1wherein the polynucleotide is DNA.
 4. The isolated and purified nucleicacid molecule of claim 1 having the nucleotide sequence of SEQ ID NO:5.5. An expression vector for expression of a mammalian histamine H4receptor in a recombinant host, wherein said vector contains a nucleicacid sequence encoding a murine histamine H4 receptor protein having theamino acid sequence of SEQ ID NO:8.
 6. The expression vector of claim 5,wherein the expression vector contains a nucleic acid molecule havingthe nucleotide sequence of SEQ ID NO:5, and encodes a murine histamineH4 receptor protein.
 7. A process for expression of mammalian histamineH4 receptor protein in a recombinant host cell, comprising: (a)transferring the expression vector of claim 5 into isolated cells; and(b) culturing the cells of step (a) under conditions which allowexpression of the histamine H4 receptor protein from the expressionvector.
 8. An isolated cell containing a recombinantly cloned nucleicacid molecule encoding a murine histamine H4 receptor protein having theamino acid sequence of SEQ ID NO:8.
 9. The isolated cell of claim 8,wherein said nucleic acid molecule has the nucleotide sequence SEQ IDNO:5.
 10. An isolated histamine H4 receptor encoded by the nucleic acidmolecule of claim
 1. 11. The histamine H4 receptor according to claim10, having the amino acid sequence SEQ ID NO:8.